The expression of a gene is regulated, at least partly, by cellular processes involved in transcription. During transcription, a single strand RNA, complementary to DNA, sequence is formed by the action of RNA polymerases. The start of transcription in eukaryotic cells is regulated by complex interaction between cis-active DNA motives located within the gene to be transcribed and trans-active protein factors. Among cis-active regulatory regions are the DNA sequences known as promoters, to which RNA polymerase primarily binds, directly or indirectly. For the purpose of the present invention, the term “promoter” refers to the specific DNA sequences—usually “upstream” (5′) of the coding region of a structural gene—that controls the expression of this coding region due to its ability to provide a recognition site for the RNA polymerase and/or other necessary factors for initiating transcription, as well as defining the correct location for gene transcription.
Typically, a promoter sequence comprises a “TATA box” and an upstream activating region. This TATA box is responsible for defining the location of transcription start site, normally approximately 25 base pairs towards the start of the coding sequence of the gene (3′).
These promoters are basically divided into inducible and non-inducible (also termed constitutive) types. An inducible promoter is one capable of activating (directly or indirectly) the transcription of one or more DNA sequences or genes in response to a determined inducer. When there is no inducer, the DNA sequences or genes are not transcribed. This inducer may be a chemical component (described in patent document WO9519443, for example), some form of stress of physiological origin (such as in the case of wounds, described in patent document U.S. Pat. No. 6,677,505, for example), or an endogenous compound generated in response to changes in plant development.
There are many tissue-specific promoters described in plants, such as the case of specific expression in seed (WO8903887), tubercle (as mentioned in patent application US20030175783, Keil et al., 1989 EMBO J. 8: 1323:1330), leaves (as mentioned in patent application US20030175783, Hudspeth et al., 1989 Plant Mol. Biol. 12:579-589), fruit (Edwards & Coruzzi (1990) Annu. Rev. Genet. 24, 275 to 303 and U.S. Pat. No. 5,753,475), stem (as mentioned in patent application US20030175783, Keller et al., 1988 EMBO J. 7: 3625-3633), vascular tissue (as mentioned in patent application US20030175783, Peleman et al., 1989 Gene 84: 359-369 and Schmülling et al. (1989) Plant Cell 1, 665-670), root (US20060143735 as mentioned in patent application US20030175783, Keller et al., 1989 Genes Devel. 3:1639-1646), stamen (WO8910396, WO9213956), specific promoters for the dehiscence zone (WO9713865) and meristem (Ito et al. (1994) Plant Molecular Biology, 24, 863 to 878).
On the other hand, constitutive promoters are capable of conducting the expression of DNA sequences throughout the development of a plant and lack specificity as to the site of sequence expression. Therefore, expression occurs in a large variety of cells and tissues of the plant. Despite this, the term “constitutive” does not imply that the sequence is expressed in similar levels in all plant cells.
Recombination techniques make it possible to trigger the transcription start site of a nucleotide sequence of interest—such as a heterologous or non-natural sequence—in a plant host cell.
The promoters that drive constitutive expression of the genes under their control may be used, for example, to select transformed plant cells, to express a selection marker gene in transgenic plants, in generating plant cells resistant to antibiotics or for creating herbicide or insecticide tolerant plants and pathogen stress resistant plants, since the products of the genes controlled by these promoters are present in all parts of the plant.
Exogenous genes of agronomic, medical or other interest may be expressed in a variety of plants, for example, to generate heterologous recombinant proteins and for the generation of plants that contain mammal polypeptides. The quantities of expression patterns—both in terms of time and space—of endogenous plant genes may also be advantageously altered with the help of constitutively active promoters.
The first promoters used for the expression of genes in plants were of viral or bacterial origin, for example, bacteria from Agrobacterium genus. Both viral and bacterial systems carry advantages in the case of heterologous expression in plants since this character constitutes the basis of their infection mechanisms. Many of these promoters have being widely used for expressing proteins of interest in the production of genetically modified plants.
Apart from Agrobacterium T-DNA derived promoters, such as those responsible for synthase of manopine (mas), octopine (ocs) and nopaline (nos), there are also virus derived promoters, with the most commonly used being CaMV35S, which corresponds to the cauliflower mosaic virus 35S promoter fragment. Later, this same promoter had its regulatory region duplicated and fused to an enhancer sequence of the alfalfa mosaic virus, generating a highly efficient recombinant plant promoter for inducing expression of coding sequences associated to it.
Other constitutive promoters of viral origin are, for example, the scrofularia mosaic virus promoter (PI1101063-0), of the badnavirus that infects the Australian banana (U.S. Pat. No. 6,391,639) and the promoter of the sugar-cane bacilliform virus (U.S. Pat. No. 6,489,462). However, viral and Agrobacterium promoters present problems related to their regulatory capacity and may be particularly unstable and suitable to horizontal gene transfer and gene recombination, which tends to highlight the importance of seeking promoters of plant origin.
Typical constitutive promoters of plants are, for example, the coffee alpha tubulin promoter (U.S. Pat. No. 6,441,273), the A. thaliana trehalose 6-phosphate synthase protein promoter (US20020115850), maize actin-2, enolase, Gos-2 and L41 promoters (U.S. Pat. No. 6,670,467), Beta vulgaris V-ATPase promoter (PI0013537-2), Brassica hsp80 promoter (PI9300296-3).
Despite that many of these plant promoters demonstrate a strong expression, data for the promoters presented herein basically refers to a qualitative analysis. However, the present invention further presents quantitative data obtained by means of fluorimetric tests that upon analysis demonstrated its high expression capacity.
The promoters described above were aligned with the present invention and showed no significant sequence identity between them. The coffee alpha tubulin promoter obtained the best alignment with an identity of 44.3%. This alignment may be performed using softwares available on the internet, with one being the BLASTN provided on the National Centre for Biotechnology Information/NCBI page: (ncbi.nlm.nih.gov.
The A. thaliana trehalose 6-phosphate synthase protein promoter (US20020115850) shows a reduction in activity in root during plant development. The present invention, however, presents high expression throughout plant tissues of transgenic Arabidopsis over 3 months, showing that uceA1.7 presents expression capacity in high levels, not only during development stages but also in mature plants, which constitutes great technological potential considering that most plants are attacked by insect-pests throughout plant's life.
It is desirable that whenever possible the available promoters enabling expression of selection genes and resistance genes present strong and uniform constitutive activity, when possible, throughout plant tissues or cell types that, furthermore, present even greater activity or are not inhibited under stress conditions.
Even if the above mentioned promoters are considered as being constitutive, time and spatial expression patterns are different rendering them inappropriate for determined purposes. This makes the search and study for other plant promoters crucial. Furthermore, high expression levels are needed to increase the levels of the protein product of interest required for certain purposes in the generation of genetically modified plants. High protein expression levels help in the generation of plants by presenting commercially important phenotype properties, such as resistance to insect-pests and diseases, environmental stress tolerance (e.g. drought, high temperatures, cold, light intensity, photoperiod and chemicals, amongst others), improved quality (e.g. high fruit yield, extended life cycle, size and color uniformity, high sugar content, high vitamin A and C and reduced acidity, amongst others).
Promoters may become more effective when isolated from the same species of the transgenic plant generated. The expression of β-glucuronidase (GUS) controlled by rice actin (Act1) promoter in protoplasts of transformed rice was approximately 6 times greater than the expression controlled by the maize constitutive promoter alcohol dehydrogenase (Adh1) (U.S. Pat. No. 658,701). Therefore, apart from being used as constitutive promoters for various plant species, the present invention advances a promoter having great advantages concerning the production of transgenic cotton plants.
Ubiquitin is one of the more conserved proteins in eukaryotes. One of the physiological functions of ubiquitin is to conjugate with a target protein as a recognition signal for protein degradation. Selective degradation of abnormal proteins is performed on many short lived regulatory proteins, including cell cycle proteins, cellular growth modulators and transcription factors. In more complex organisms, ubiquitin has been encoded by two small gene families termed “polyabiquitin genes” and “ubiquitin fusion genes”. Polyubiquitin genes comprise a tandem repetition of 228 bp head to tail, with each repetition encoding 76 amino acids of an ubiquitin monomer. The number of tandem repetitions indicated variations in the genes within the genomes and between organisms, ranging from 3 in Dictostylium to approximately 50 in Trypanosoma cruzi. On the other hand, the ubiquitin fusion gene family encodes a simple repetition fused with one or two polypeptides having 52 or 76-80 amino acids each (Callis et al., “Ubiquitin and Ubiquitin Genes in Higher Plants”, Oxford Surveys of Plant Molecular & Cell Biology, (1989), vol. 6, pp. 1-30). Studies of ubiquitin genes in different plants show that ubiquitin genes are expressed in all tissues; however, the differential expression of ubiquitin genes can also be observed in the ubiquitin gene family. Each tandem repetition or ubiquitin gene may be differentially expressed in cells or tissues.
Ubiquitin gene promoters have demonstrated the ability to conduct the expression of genes—usually GUS or chloramphenicol acetyltransferase (CAT)—in transformed cells or plants. Such promoters have been isolated from Arabidopsis (Callis et al., “Ubiquitin Extension Proteins of A. thaliana”, The Journal of Biological Chemistry (1990), vol. 265, n. 21, pp. 12486-12493); sunflower (Binet et al., “Analysis of a sunflower polyubiquitin promoter by transient expression” (1991) Plant Science, vol 79, pp. 87-94); tobacco (Genschick et al., “Structure and promoter activity of a stress and developmentally regulated polyubiquitin encoding gene of Nicotiana tabacum”, Gene, (1994) vol. 148, pp. 195-202); maize (US20030066108; U.S. Pat. No. 6,020,190; U.S. Pat. No. 5,614,399; U.S. Pat. No. 5,510,474; Christensen et al., “Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation”, Plant Molecular Biology, (1992) vol. 18, pp. 675-689); rice (U.S. Pat. No. 6,528,701); sugar-cane (U.S. Pat. No. 6,706,948); celery (WO2003102198); Pine and eucalyptus (PI0309870-2). These promoters were aligned with the present invention using BLASTN and showed no similarity. Patent documents US20030066108; U.S. Pat. No. 6,020,190; U.S. Pat. No. 5,614,399 and U.S. Pat. No. 5,510,474 describe engineered versions of the maize ubiquitin promoter used to increase expression levels compared to the expression levels of the native ubiquitin promoter. The invention reports that the promoter regulates the expression of maize polyubiquitin gene containing 7 tandem repetitions. The expression of this ubiquitin gene proved constitutive at 25° C. and was heat induced at 42° C. This promoter was used successfully to transform monocots other than maize, including wheat, barley and rice. However, the promoter of the present invention proved to be an excellent constitutive promoter and does not undergo any change in expression levels when submitted to different temperatures as well as presenting high expression throughout the plant cycle.
Cotton (Gossypium spp) is one of the most important cultures in the world, and is considered to be the most important of all textile fibres. Brazil holds a prominent position amongst the world's main cotton producers. However, the increase of production costs is influenced by agricultural problems, such as insect pest control, which represents approximately 25% of total production costs.
The production of genetically modified plants expressing proteins conferring resistance to the plant has been used with great success in insect-pests control. However, it is necessary to control and target the expression of these entomotoxic proteins in order to obtain these plants. The choice of the promoters that conduct expression is very important for obtaining transgenic plants affording adequate protein levels conferring insect resistance to plants. However, there are few effective promoters for expressing cotton plants available on the market at present, and none that demonstrate superior expression efficiency compared to the promoter of the present invention.